TGF-.beta. is the prototype of a family of structurally related cytokines capable of inducing diverse cellular responses, including apoptosis, differentiation, and cell cycle arrest. Cancer cells often lose responsiveness to TGF-.beta., an event which apparently affords a growth advantage and favors tumor progression. The fact that components of the TGF-.beta. signaling pathway are mutated in cancers provides compelling support for this idea. In this regard, mutations in the TGF-.beta. receptor type II gene are prevalent in cancers with mismatch repair deficiency, Smad4 mutations commonly occur in pancreatic carcinomas, and Smad4 and Smad2 mutations are found in a subset of colorectal cancers.
Two types of TGF-.beta. receptor (TGF-.beta.R), each containing an intracellular serine/threonine kinase domain, act in concert to activate signaling. TGF-.beta.RII binds TGF-.beta. and subsequently phosphorylates TGF-.beta.RI. The activated RI receptor then propagates the signal by phosphorylating intracellular targets, the most important of which in mammalian cells appear to be the Smad proteins. The Smad genes are homologues of Drosophila and C. elegans genes identified as critical mediators of TGF-.beta.-like signaling. In vertebrate cells, Smads 1, 2, 3 and 5 are phosphorylated by activated RI receptors at a conserved SSXS motif located at their carboxyl termini, whereupon they translocate from the cell membrane or cytoplasm to the nucleus. Smad4 (a.k.a. DPC4) lacks the signature phosphorylation site at its C-terminus and has not been found to associate with the TGF-.beta.R complex. Instead, Smad4 forms heteromeric complexes with other Smads after ligand stimulation and may be important for their translocation to the nucleus.
In mammalian cells, Smad2 and Smad3 are phosphorylated upon TGF-.beta. stimulation while Smad1 is phosphorylated in response to BMP. Once heterodimerized with Smad4 and transported to the nucleus, the mechanisms through which Smad proteins propagate TGF-.beta. signaling are unclear. When expressed as fusion proteins with Gal4, the carboxyl-terminal domains of Smad proteins confer transcriptional activation to reporters containing Gal4-binding sequences. Smad proteins can complex with Xenopus FAST-1, a protein which possesses a DNA-binding domain. These observations have suggested that a complex composed of Smad proteins and FAST-1 may transcriptionally activate promoters containing DNA sequences recognized by the latter protein. More recently, it has been shown that the amino-terminal domain of Drosophila Mad can itself bind to elements within the vestigal (Vg) promoter, suggesting a sequence-specific binding function that might directly account for the biologic activity of the Mad protein. However, the full length Drosophila Mad protein did not exhibit DNA-binding activity, raising questions about the role of this binding in vivo. In another study, human Smad4, but not Smad3, was shown to bind to sequences from p3TP-lux, a reporter commonly used to assess TGF-.beta. responses. Interestingly, the sequences responsible for binding of Drosophila Mad to the Vg promoter bore little resemblance to the p3TP-lux elements that bound to Smad4. Additionally, the sequences within p3TP-lux that bound to Smad4 were completely dispensable for TGF-.beta.-dependent activation of this reporter.